Abstract The migrating growth cone at the distal axon tip senses and responds to guidance information. Growth cones display dynamic lamellipodial and filopodial protrusions that are involved in outgrowth and guidance. The aim of these studies is to use C. elegans to understand the cytoskeletal signaling networks that link axon guidance signals to changes in growth cone morphology and outgrowth via the actin cytoskeleton. Results from the previous grant period showed that three actin-regulatory systems, Arp2/3, UNC-115/abLIM, and UNC-34/Enabled, redundantly control axon pathfinding. The Arp2/3 complex and UNC-115/abLIM act redundantly downstream of Rac GTPases and in parallel to UNC-34/Enabled. Each of these molecules controlled the formation of growth cone filopodia, possibly explaining axon pathfinding defects in mutants. That multiple actin regulatory systems contribute to axon guidance and the formation of growth cone filopodia explains the genetic redundancy often observed in studies of axon guidance. In order to understand the molecular events in the growth cone that regulate actin structure, it will be important to understand how these individual actin regulatory networks function and how they interact in growth cone outgrowth and guidance. In this proposal, genetic, molecular, and in vivo cellular imaging approaches will be used to dissect the roles of cytoskeletal signaling pathways in growth cone morphology and outgrowth. Many pathways and molecules have been identified that affect axon pathfinding. These experiments move beyond the "gene by gene" approach to studying axon pathfinding and instead are designed to understand how molecules relate to one another in pathways and networks to control axon pathfinding and growth cone morphology. To complement the genetic analyses, in vivo cellular imaging studies are designed to understand the unique or overlapping contributions of the often genetically-redundant pathways to growth cone morphogenesis in a developmental context. The focus of the proposal is the idea that the CDC-42 GTPase acts upstream of Rac GTPases in axon pathfinding. Experiments to characterize molecules that regulate CDC-42 and those that might link CDC-42 to Rac signaling are proposed. The first and second aims aim test the idea that CDC-42 acts upstream of Rac GTPases in axon pathfinding. The third aim is to probe the role of the MIG-15 NIK kinase and RACK-1/Receptor for activated C kinase and their interaction with UNC-115/abLIM downstream of CDC-42 in a Rac-independent pathway. The fourth aim integrates the first three and is to characterize the effects of these pathways on growth cone filopodia formation and morphology during outgrowth. The results of these experiments will significantly contribute to the goal of understanding the cytoskeletal signaling networks involved in growth cone morphology and will begin to address the cellular roles of these distinct pathways in axon development.